For membrane proteins, such a method is known from the article “Refolding of Escherichia coli produced membrane protein inclusion bodies immobilised by nickel chelating chromatography”, Rogl et al. in FEBS Letters 432 (1998) 21–26.
A method for refolding of receptor protein is known from the article “Expression of an Olfactory Receptor in Escherichia coli: Purification, Reconstitution, and Ligand Binding” by Kiefer et al. in Biochemistry 35 (1996) 16077–16084.
Both publications are based on the problem that membrane proteins, to which receptors also belong, can be produced in large quantities with the help of expression vectors in bacteria, but that the protein produced, however, is not active. The protein is, namely, not integrated into the membrane, but, first, is present in a denatured state and has to be refolded into the native or active structure. The aggregates of “inactive” protein are designated in English literature as inclusion bodies.
For the membrane proteins Toc75 and LHCP, Rogl et al. describe a method in which N-Lauroylsarcosine is used as first detergent and Triton X-100® is used as second detergent. By exchanging the chaotrope for the mild detergent, refolding of the aggregated protein was induced.
According to Kiefer et al., a G-protein-coupled olfactory receptor was transformed into the active structure during the binding onto a nickel column by detergent exchange from N-Lauroylsarcosine to digitonin.
In both cases, it could be shown that the aggregated protein first existing in the form of inclusion bodies was, first, solubilized in a denaturing detergent and, then, by the detergent exchange described, transformed into its active structure, which was verified by corresponding binding measurements.
There is a great scientific and commercial interest in membrane proteins, in particular in receptors in native or active form, since membrane proteins are components of all biological membranes and impart to the specificity of different cellular membranes, they are particularly responsible for the exchange of substances and signals.
The specific recognition of a chemical compound by the corresponding receptor has e.g. the consequence that the target cell changes its physiological state. That is why receptors are the most important target molecules for drugs, approximately ¾ of all commercially available pharmaceuticals act on receptors, most of which, again, act on so-called G-protein-coupled receptors, which have in the human genome several hundreds of representatives.
For the development of specific antibodies, of drugs etc. it is, in view of the above, most desirable to have membrane proteins, in particular receptors in active or native structure available in large quantities. Since these proteins occur, in tissue, only in very small concentrations, it is necessary to use a system for recombinant over-expression of membrane proteins and receptors. For this purpose, on the one hand, in eukaryotic cells (cells of mammals or insects), functional protein can be produced, however, the systems are expensive, and the expression rates are low, which is also disadvantageous. Functional protein can be obtained via bacterial expression as well, the expression rate, however, is even lower than in eukaryotic expression.
In view of the above, the two publications mentioned at the outset describe methods, in which the protein is expressed in the inner part of the cell, where it, however, aggregates, and hence is not functionally available. The advantage of this method is that very large quantities of protein can be produced, Kiefer et al. report that up to 10% of cell protein and, thus, 100–10,000 times more protein than with other expression systems can be produced. The inclusion bodies produced in that way, which Rogl et al. have also reported about, must then first of all, be solubilized and, via the exchange of detergents already described at the outset, be transformed into their native or active structure.
Of course, commercial interest is not only directed to membrane proteins and receptors in their naturally existing sequence, rather, also partial sequences, homologous sequences, mutated sequences or derived sequences of membrane proteins and receptors are an object of this invention, as they allow, depending on functionality, not only insights into the structure of membrane proteins and receptors, but also a rational drug design.
In this context it should be mentioned that the DNA sequence of many receptors is known, such sequences are contained in the EMBL database. As these DNA sequences, in most cases, do not contain introns, the coding sequence can be produced by PCR from genomic DNA or by RT-PCR from mRNA. This DNA can then be cloned into a corresponding expression vector.
However, the structure of the translation product is unknown, so that providing proteins which are an object of the invention in sufficient quantity allows crystallization experiments etc. to further elucidate the structure.
It should also be mentioned that receptors expressed in eukaryotic and in bacteria can be distinguished by glycosylation. G-protein-coupled receptors, namely, possess on the N-terminus one or more glycosylation sites, which are modified in the endoplasmic reticulum or later in the Golgi apparatus with an oligosaccharide. Bacteria, in contrast, do not modify these sequences.
By treating a portion of the protein with N-glycosidase F or N-glycosidase A, the saccharide can be cleaved off, so that on an SDS gel a different extend of migration of the protein can be distinguished before and after this treatment, if the protein was expressed in eukaryotic cells. For bacterially expressed protein, no differences in the extend of migration can be distinguished.
Although the methods for the production of membrane protein or receptor protein that are described in the publications mentioned above lead to active structures, the methods described, according to the knowledge of the inventors of the present application here submitted, are insofar not satisfying, as the yield is low and the method is poorly reproducible.